This invention lies in the field of recombinant DNA technology and is directed at a cell having a certain function in a process, containing recombinant DNA encoding at least one enzyme. The invention is directed especially at a cell having a function in the field of food processing and also at cells with a function in processes in which a cellulose-containing raw material is used, such as processes for preparing beer, paper, starch, gluten etc, and processes for decomposing cellulose-containing waste such as agricultural waste, waste from paper rills etc.
In particular the invention is directed at cells having a function in the process of fermentation, more especially at cells with a function in the process of preparing bakery products.
The cell according to the invention is characterized in that the cell becomes polyfunctional for the process in which it has a function, upon expression of the recombinant DNA encoding at least one enzyme. In the case of a fermentation process for example, such as the preparation of bread, yeast is used as a cell with a particular function in said process. A yeast cell according to the invention does not only have its normal function, i.c. a function that a yeast lacking the recombinant DNA can also carry out, but also has another function in said process of bread preparation. An example of such an additional function is the expression and secretion of a bread improving enzyme.
The present invention is directed in particular at a cell with a function in the preparation of bakery products. Cells containing recombinant DNA encoding enzymes selected from the group of enzymes with amylolytic and/or hemicellulolytic and/or cellulolytic activity are suitable.
The invention is also directed at a process for the production of at least one enzyme by a polyfunctional cell as described above comprising culturing such a polyfunctional cell in a suitable nutrient medium and optionally isolating the resulting enzyme form. In such a process, said enzyme is preferably selected from the group of enzymes with amylolytic and/or hemicellulolytic and/or cellulolytic activity. A suitable medium for carrying out the process according to the invention can consist of the medium in which the process for which the cell is polyfunctional is carried out. In the process of the preparation of a bakery product for example said medium can be the dough that it to be baked. Naturally the other usual media for culturing cells can also be used. The choice of media will depend on whether the enzyme is to be used in situ or has to be isolated. In some cases it will suffice to use the medium containing said enzyme and in other cases the enzyme will have to be isolated from the medium.
The invention is also directed at an enzyme encoded by the recombinant DNA in said polyfunctional cell, whereby said enzyme is obtainable from such a polyfunctional cell via the afore mentioned process for producing an enzyme. The invention is further directed at the use of such a polyfunctional cell or such an enzyme, for example in the processes described above, such as food processing and processes using a cellulose containing raw material, preferably in a process for the preparation of a bakery product.
Flour, yeast, water and salt are the basic ingredients of bread and other bakery products. For centuries materials having a positive effect on the manageability of the dough or the quality of the baked product have been added in the manufacture of bread and similar bakery products, for the sake of convenience further referred to as bread making. Said additives, referred to as xe2x80x9cbread improversxe2x80x9d, contain enzymes from malt or of microbial origin which play an important part in the different phases of bread making, namely, the preparation of the bread batter, fermentation, baking, and storage of the bread product.
One of the relevant characteristics of bread that is influenced by adding specific enzymes is the so-called bread volume. In order to obtain a high bread volume in practice compositions containing cellulolytic, hemicellulolytic and/or amylolytic enzymes are added. The commercially available compositions of microbial origin, mostly originating from a fungus o; one of the genera Aspergillus and Trichoderma, are substantially unpurified complex mixtures of different enzyme activities, whereby it is not exactly known which enzymes are present in the composition and which have a bread improving activity. This lack of knowledge impedes further bread improvement and especially impedes the control of the different dough processing and bread properties, such as the bread volume.
Further investigation into the process of preparation of bakery products resulted in the discovery that, in addition to xcex1-amylase, at least a xylanase enzyme is also of importance for the bread volume. A xylanase is an enzyme that catalyzes the degradation of xylans occurring in the pentosan part of starch xe2x80x9ctailingsxe2x80x9d. The term xe2x80x9ctailingsxe2x80x9d is directed at a fraction of, e.g., wheat starch consisting of water-insoluble hemicellulose (pentosans and arabinoxylans) and damaged starch. This fraction is formed as the intermediate or top layer of the starch pellet during centrifugation of a dough suspension obtained by washing dough to remove the gluten fraction.
Different xylanases have already been described in the literature, including xylanases of the bacterial species Bacillus pumilus (Panbangred et al., Mol. Gen. Genet. 192, 335-341, 1983, and Fukusaki et al., FEBS Lett. 321, 197-201, 1984), Bacillus subtilis (Paice et al., Arch. Microbiol. 144, 201-206, 1986), and Bacillus circulans (Yang et al., Nucl. Acids Res. 16, 7187, 1988), of the yeast Aureobasidium (Leathers, Biotech. Lett. 12, 775-780, 1988) and of the fungus Aspergillus niger (Fournier et al., Biotechnology and Bioengineering 27, 539-546, 1985).
It is known from European patent application EP-A-0338452 that the properties of dough and the quality of bread can be improved by adding different enzyme compositions to the dough, including an enzyme composition having hemicellulose degrading or xylanase activity, the origin of which is not further specified. Such a hemicellulolytic enzyme composition is a relatively undefined enzyme mixture which may contain different hemicellulolytic enzymes having various effects on the dough and bread properties. The presence of xylanases having bread improving activity to a smaller or greater extent is the coincidental result of the manner in which the enzyme composition that is intended as a bread improver has been obtained. A controlled further optimization of bread improvers, however, was not possible due to the lack of the required knowledge and of suitable recombinant DNA constructs encoding a xylanase having broad improving activity that could be used for a high production of such a xylanase.
For the purpose of this invention, xe2x80x9cbread improving activityxe2x80x9d is generally taken to mean a favourable effect on any property of the prepared bakery product (including bread) or of the dough from which the bakery or bread product is made, and is particularly taken to mean a favourable effect on the bread volume.
The investigation on which the invention is based has extended to the identification and cloning of a gene (xylA) encoding an enzyme, xylanase having bread improving activity, originating from a fungus of the species Aspergillus niger var. awamori, as well as to the transformation of different species of host cells in such a manner that the gene is expressed or can be expressed in said host cells. The invention, however, comprises all xylanase genes originating from fungi and especially from fungal strains from the same genus and therefore the invention is not limited to the actually cloned gene.
The present invention is therefore also directed at recombinant DNA material comprising DNA with a nucleotide sequence encoding at least a ripening form of a xylanase of fungal origin.
The term xe2x80x9cripening formxe2x80x9d refers to the different forms in which the enzyme may occur after expression of the associated gene. More in particular, it refers to both the naturally and the not naturally occurring prepro-, pre- and pro-forms and to the ultimate mature form of the enzyme resulting after cleavage of a xe2x80x9cleaderxe2x80x9d peptide.
More in particular, the invention, relates to recombinant DNA material comprising DNA with a nucleotide sequence encoding at least a ripening form of a xylanase of Aspergillus origin.
Preferably, this aspect of the invention is concerned with recombinant DNA material comprising DNA with a nucleotide sequence encoding a ripening form of a xylanase of Aspergillus niger origin, especially of Aspergillus niger var. awamori origin.
A preferred embodiment of this aspect of the invention is recombinant DNA material comprising DNA with a nucleotide sequence encoding at least a ripening form of xylanase with an amino acid sequence as shown in FIG. 1 (SEQ ID NO:7), and more in particular recombinant DNA material comprising DNA with a nucleotide sequence encoding a ripening form of xylanase, as shown in FIG. 1 (SEQ ID NO:7). The invention is also directed at recombinant DNA material comprising DNA with a nucleotide sequence encoding at least a ripening form of xylanase with a nucleotide sequence that is equivalent to the nucleotide sequence of FIG. 1 (SEQ ID NO:7) with deletions, insertions or alterations in comparison to the nucleotide sequence of FIG. 1 (SEQ ID NO:7) such that the nucleotide sequence with deletions, insertions or alterations corresponds either to the amino acid sequence as shown in FIG. 1 (SEQ ID NO:7) or to those parts of the amino acid sequence of FIG. 1 (SEQ ID NO:7) essential for an active ripening form of xylanase such as mature xylanase or active pre(pro) xylanase or the nucleotide sequence with deletions, insertions or alterations has a complementary strand capable of hybridizing under hybridizing conditions to the nucleotide sequence of FIG. 1 (SEQ ID NO:7).
The the recombinant DNA according to the invention contains at least a sequence encoding the fungal, in particular the Aspergillus xylanase ripening form. In addition, the recombinant DNA may contain many other types of information, such as regulating sequences (especially a transcription promoter) and a vector part usually provided with one or more marker genes. These other types of information will often be connect d with the selected host. Thus, for instance, the vector, the marker genes and the regulating sequences will be selected depending on the selected host.
The recombinant DNA encoding at least a ripening xylanase of fungal origin, however, may also contain other genes to be expressed in the selected host. Such a gene may advantageously encode at least one other enzyme, wherein said other enzyme has amylolytic and/or hemicellulolytic and/or cellulolytic activity.
Another aspect of the invention is a cell containing genetic material derived from recombinant DNA material according to the invention as defined above, and more in particular such a cell capable of expression of at least the xylanase ripening form encoded on said recombinant DNA material. A preference exists for such a cell that is also a polyfunctional cell according to the invention and more especially for such a polyfunctional cell capable of expressing the recombinant DNA material encoding a ripening form of xylanase of fungal origin under conditions present in raw material during preparation of a bakery product.
Both a polyfunctional cell containing recombinant DNA encoding at least one enzyme according to the invention, and a cell containing recombinant DNA material encoding a ripening form of xylanase of fungal origin according to the invention (as well as the combination thereof) may either be a cell which is itself the direct result of gene manipulation or be a cell originating in any manner from a cell that has been transformed by such gene manipulation. The invention further extends to both live cells and cells that are no longer alive.
In principle the invention knows no special limitations with respect to the nature of the cells, whereby those cells capable of expression of a xylanase ripening form of fungal origin are preferred. However the cells are preferably selected from the group consisting of bacterial cells, fungal cells, yeast cells, and plant cells.
Preferred examples of eminently suited host cells are
(a) fungal cells of one of the genera Aspergillus and Trichoderma, in particular fungal cells of one of the species Aspergillus niger var. niger, Aspergillus niger var. awamori, Aspergillus nidulans, Aspergillus oryzae Trichoderma reisei and Trichoderma viride; 
(b) yeast cells of one of the genera Saccharomyces, Kluyveromyces, Hansenula and Pichia, in particular yeast cells of one of the species Saccharomyces cerevisiae, Saccharomyces carlbergensis, Kluyveromyces lactis, Kluyveromyces marxianus, Hansenula polymorpha and Pichia I storis; 
(c) plant cells of a plant genus selected from the group consisting of wheat, barley, oats, maize, pea, potato and tobacco, such as plant cells of one of the species Solanum tuberosum and Nicotiana tabacum; and
(d) bacterial cells of one of the bacterial genera Bacillus, Lactobacillus and Streptococcus, such as bacteria of the species Bacillus subtilis. 
Cells according to the invention as defined above (polyfunctional and/or simply containing recombinant DNA encoding a ripening form of xylanase of fungal origin) may be important as agents for multiplying the recombinant DNA or as agents for producing at least one enzyme encoded on said recombinant DNA, such as the ripening form of xylanase.
In the case of enzyme production it is possible to use the cell to produce enzyme and either isolate the enzyme from the culturing medium or use the medium containing the enzyme after removal of the cells as such, or in the case of the polyfunctional cells to use the cells themselves to produce the enzyme in situ in the process for which they are poly-functional.
A direct use of the cells themselves is possible, e.g., if the host strain can be admitted without objection, in the production of foodstuffs as is the case for various fungal, yeast, plant, and bacterial species. In connection with bread making the yeast strains that are genetically manipulated in accordance with the present invention can for example be used directly.
Partly depending on the selected host the gene encoding xylanase will be used, either with or without introns occurring in said gene, either with its own transcription termination signals or originating from another gene, and either with its own leader sequence or with a signal sequence originating from another gene. For transformation of yeast, such as Saccharomyces cerevisiae (baker""s yeast), it is preferable that the introns are removed and that the own leader sequence is replaced by a signal sequence suitable for yeast, such as the signal sequence of the invertase gene, ensuring correct processing and secretion of the mature protein.
The removal of introns is necessary upon transformation of bacteria, such as Bacillus subtilis. In this case e.g. the xcex1-amylase signal sequence can be used as signal sequence.
Suitable transformation methods and suitable expression vectors provided with, e.g., a suitable transcription promoter, suitable transcription termination signals, and suitable marker genes for selecting transformed cells are already known for many organisms, including different bacterial, yeast, fungal, and plant species. Reference may be made for yeast for example to Tajima et al., Yeast 1, 67-77, 1985, which shows expression of a foreign gene under control of the GAL7 promoter inducible by galactose in yeast, and for Bacillus subtilis for example to EP-A-0 157 441, describing a plasmid pMS48 containing the SPO2 promoter as an expression vector. For other possibilities in these and other organisms reference is made to the general literature.
In another aspect the present invention consists of a ripening form of a xylanase of a fungus, in particular of Aspergillus origin, obtained by expression of recombinant DNA material according to the invention, as defined above. Herein, special preference is given to a mature xylanase with an amino acid sequence as illustrated in FIG. 1, as well as to a pre(pro)-xylanase with an amino acid sequence as illustrated in FIG. 1 (SEQ ID Nos: 7 and 8) and to any amino acid sequence of an active equivalent form of xylanase comprising the amino acids of the sequence of FIG. 1 which are essential for xylanase activity. The invention is therefore directed at an amino acid structure leading to a tertiary enzyme structure with the same enzyme activity as the enzyme with the sequence of FIG. 1.
Yet another aspect of the invention consists of a process for producing a ripening form of a xylanase of a fungus, in particular of Aspergillus origin, comprising culturing a polyfunctional cell capable of expressing a xylanase ripening form and or a cell capable of expressing the recombinant DNA material according to the invention encoding a ripening form of xylanase of fungal origin in a suitable nutrient medium, and optionally isolating the resulting xylanase ripening form. The term xe2x80x9cisolating the resulting xylanase ripening formxe2x80x9d also comprises a partial purification in which an enzyme composition is recovered comprising the relevant xylanase.
Further aspects of the present invention are a bread improver composition comprising an enzyme selected from the group of enzymes with amylolytic and/or hemicellulolytic and/or cellulolytic activity such as a ripening form of xylanase, in particular a mature xylanase of a fungus, especially of Aspergillus origin, whereby said enzyme is obtainable from a polyfunctional cell according to the invention and/or from expression of recombinant DNA according to the invention encoding a ripening form of a xylanase of fungal origin and a bread improver composition comprising a polyfunctional cell according to the invention; a flour and dough composition comprising an enzyme selected from the group of enzymes with amylolytic and/or hemicellulolytic and/or cellulolytic activity such as a ripening form of xylanase, in particular a mature xylanase of a fungus, especially of Aspergillus origin, whereby said enzyme is obtainable from a polyfunctional cell according to the invention and/or from expression of recombinant DNA according to the invention encoding a ripening form of a xylanase of fungal origin; a flour and dough composition comprising a polyfunctional cell according to the invention; a bakery product obtained using such flour or dough compositions as described above; and a process for the preparation of a bakery product, using such flour or dough compositions especially in which a mature xylanase of a fungus, in particular of Aspergillus origin is included.
The invention, however, also extends to other uses of fungal xylanases, such as use within the scope of beer making, particularly the preparation of beers on the basis of wheat, in order to improve filterability, use in the paper-making industry to reduce water absorption by the paper material, use in the treatment of agricultural waste, etc.
The invention will now be elucidated by means of an extensive description of the identification, cloning and expression of a xylanase suitable as a bread improver. In the experimental work described in the examples the fungal strain Aspergillus niger var. awamori CBS 115.52 (ATCC 11358) is used as a source for the xylanase. According to investigations carried out by the inventors, said strain, after induction with wheat bran, is capable of producing a xylanase having bread improving properties, while the culture medium exhibits an xcex1-amylase activity, a low glucanase activity and a low protease activity under these induction conditions. The amount of xylanase produced by the wild-type strain, however, is too low for use in a commercial process. For this reason the invention also provides gene manipulations enabling a biotechnological production of the xylanase on a commercial scale.
The conducted experimental work comprises the isolation of the gene encoding a xylanase enzyme (the xylA gene) from a gene library of chromosomal Aspergillus niger var. awamori DNA made in a xcex vector. For said isolation a probe was made with a composition derived from the N-terminal amino acid sequence of the purified mature protein as determined by the inventors. By means of this probe a number of xcex clones were isolated which possibly contained the gene. A DNA fragment from these positive xcex clones was subcloned. Subsequently, the DNA sequence of part of the cloned chromosomal DNA fragment was determined. By means of these results and those of mRNA analysis, the length of the xylA gene, the length of the mRNA, and the presence and position of an intron have been determined. It could be derived from the data that the xylA gene encodes a protein of 211 amino acids (a pre(pro)-form) in which the mature protein of 184 amino acids is preceded by a xe2x80x9cleaderxe2x80x9d peptide of 27 residues.
Three expression vectors containing the xylanase gene including the xylA terminator have been constructed. In one of these vectors the xylA gene is preceded by its own expression signals. In the second vector the xylA expression signals (up to the ATG codon) have been replaced by the constitutive expression signals of the Aspergillus nidulans glyceraldehyde 3-phosphate dehydrogenase (gpdA) gene (see Punt et al., Gene 69, 49-57, 1988), while in the third vector the xylA gene is preceded by the inducible expression signals of the Aspergillus niger var. niger glucoamylase (glaA) gene. All the expression vectors contain the Aspergillus nidulans acetamidase (amdS) gene as selection marker as described by K. Wernars, xe2x80x9cDNA mediated transformation of the filamentous fungus Aspergillus nidulansxe2x80x9d, thesis, Landbouw Hogeschool Wageningen 1986. By means of this selection marker transformants can be obtained in which the vector, and consequently also the xylA gene, is integrated into the genome in a large number of copies.
Multicopy transformants were obtained by transformation of the Aspergillus strains A. niger var. awamori and A. niger var. niger N402 with the above mentioned expression vectors. In shaking flask experiments the production of xylanase was measured after culturing the resulting transformants in different media. The results (maximum production levels) are listed in Table A given below, in which the xylanase activity is expressed in 103 units (U) per ml. A unit is defined as the amount of enzyme which, per 1 minute, releases an amount of reducing groups from xylan equivalent to 1 mg xylose.
After induction with xylan the A. niger var. awamori and A. niger var. niger N402 xe2x80x9cxylAxe2x80x9d multicopy transformants with xylA promoter produce much more xylanase than the wild type A. niger var. awamori and A. niger var. niger strains. From this and from data obtained in the molecular analysis of the gene it can be derived that the cloned gene encodes a functional xylanase. Furthermore it is apparent from the afore mentioned that the multicopy transformants are capable of overproduction of the active enzyme. In baking tests this enzyme composition also has the desired properties.
Multicopy transformants of the host strains with the heterologous gpdA or glaA promoter are also capable of an increased production of active xylanase. In rich medium the xe2x80x9cgpdAxe2x80x9d transformants produce a clearly larger amount of xylanase than the wild type A. niger var. awamori strain. However, the production levels observed in the conducted tests are substantially lower than the level obtained in the tests with xe2x80x9cxylAxe2x80x9d multicopy transformants. After induction with starch the production levels of xe2x80x9cglaAxe2x80x9d multicopy transformants are comparable to those of xe2x80x9cxylAxe2x80x9d multicopy transformants in xylan medium.
In medium with wheat bran the best A. niger var. awamori xe2x80x9cxylAxe2x80x9d multicopy transformants produce much more xylanase than is the case in xylan medium. In this medium the best A. niger var. niger N402 xe2x80x9cxylAxe2x80x9d transformants reach a very high xylanase production level. The highest producing xe2x80x9cgpdAxe2x80x9d multicopy transformants of both A. niger var. awamori and of A. niger var. niger N402 in bran produce as much xylanase as in rich medium. In medium with wheat bran the production by A. niger var. awamori xe2x80x9cglaAxe2x80x9d transformants is lower than in starch. In this medium, however, A. niger var. niger N402 xe2x80x9cglaAxe2x80x9d transformants produce more than in starch.
The production reached by Aspergillus niger var. niger N402 transformants is higher than that of Aspergillus niger var. awamori transformants. The production level of the A. niger var. awamori transformants, however, can be further increased by using suitable A. niger var. awamori mutant strains, such as A. niger var. awamori #40, which produces clearly more xylanase than the wild type strain. The mutant A. niger var. awamori #40 has been obtained by mutagenesis of A. niger var. awamori spores and selection for xylanase production. In bran medium the xe2x80x9cxylAxe2x80x9d A. niger var. awamori #40 transformant produced 190 000 U xylanase, which is a considerable increase over the best producing A. niger var. awamori transformant.
Further experiments relate to the isolation and use of the thus produced xylanase as a bread improver (see example II) and expression experiments in a yeast strain and a bacterium (examples III and IV, respectively). While example V demonstrates the use of a polyfunctional yeast according to the invention in the preparation of bread, whereby said yeast produces xylanase during fermentation of lean bread dough.